The Genome of Melampsora larici-populina, The Poplar Leaf Rust Tree/Microbe Interactions, INRA/Nancy University

Genome Sequencing of Micro-organisms Associated to Poplar - the ectomycorrhizal Laccaria bicolor (65 Mb) (released) - the ectomycorrhizal Paxillus involutus (18 Mb) (sequencing agreed) - the endomycorrhizal Glomus intraradices (x Mb) (annotation on-going) - the rust fungus Melampsora larici-populina (100 Mb) (annotation on-going) - the ectomycorrhizal Tuber melanosporum (120 Mb) (annotation on-going) + the mycorrhiza helper Pseudomonas fluorescens BBc6R8 (7 Mb) (annotation on-going) Sequencing of the Populus Community Genome F Martin, P Lammers & G Tuskan Glomus Melampsora Laccaria Populus Tuber Pseudomonas fluorescens Martin et al, New Phytol 2004

The Melampsora Genome Consortium DOE Joint Genome Institute ( Igor Grigoriev, Harris Shapiro, Erika Lindquist, Andrea Aerts, Jeremy Schmutz & Sequencing/Assembly staffs INRA Nancy – UMR IaM ( Sébastien Duplessis, Francis Martin, Marie-Pierre Oudot-Le Secq, Emilie Tisserant, Benoît Hilselberger, Stéphane Hacquard, Pascal Frey, Fabien Halkett, Axelle Andrieu Nancy Université – UMR IaM Claire Veneault-Fourrey, Nicolas Rouhier, Eric Gelhaye, Benjamin Selles Ghent University, VIB Yao-Cheng Lin, Pierre Rouzé, Yves Van de Peer Canadian Forest Service Nicolas Feau, David Joly, Philippe Tangay, Richard Hamelin Marseille, Göttingen, Lausane Universities Pedro Coutinho, Bernard Henrissat – Ursula Kües, Hélène Niculita-Herzel Melampsora larici-populina Genome

Comparative analysis of genomes from saprotrophic, pathogenic and symbiotic basidiomycetes, e.g. the symbiotic Laccaria, the white-rot Phanerochate, the coprophylic Coprinopsis © Broad Institute © INRA saprotrophy symbiosis pathogenesis The complement of genes is very conserved in eukaryotic lineages. Evolutionary innovation in eukaryotes relies mostly on carrying different versions (alleles) of the same gene. - genes (or gene networks) specific to each functional groups? - acquisitions or loss of genes? - common genes, but specific regulatory networks?

Comparative Genomics. between biotrophic fungi. among saprotrophic, symbiotic & pathogenic basidiomycetes Melampsora larici-populina Puccinia graminis Aspergillus niger Sporobolomyces roseus Phakopsora pachyrhizi Melampsora larici-populina Dikaria Basidiomycota Uredinales Melampsoraceae

Uredospores Genomic Libraries & Sequencing 1 st Assembly 200  g HMW DNA (S Duplessis) Feb 2006 Dec 2007 Basidiomycetes, Uredinales, Melampsoracea, Melampsora larici-populina 98AG31 ab initio Annotation JGI Web Portal Melampsora larici-populina/ Populus trichocarpa © INRA strain 98AG31 dikaryotic (2 x N) (collected by P Frey) Mars 2005 CSP Proposal Submission – May 2005 CSP Proposal Approved Jan 2007 Manual curation Genome analysis June 2008 August st Genome Annotation Workshop Sequencing 2 nd Assembly

Coverage & Production Stats: LIBCOV.READSINSERT± STD BTCO2.71x577,8843,968± 346 BZIT3.15x615,7476,283± 467 BTCP0.03x7,6808,511± 766 BTCS0.89x278,77736,907± 4,627 Total6.79x1,480,088

Melampsora larici-populina Genome Melampsora larici-populina Assembly (Dec 2007) Arachne assembly - Main genome scaffold total: Main genome contig total: Main genome scaffold sequence total: MB - Main genome contig sequence total: 97.7 MB (3.4% gap) - Main genome scaffold N/L50: 27/1.1 MB - Main genome contig N/L50: 265/112.3 KB - Number of scaffolds > 50 KB:155 -% main genome in scaffolds > 50 KB: 96.5% - Main genome depth: 6.79X 2 x N Moderately polymorphic genome: Variation within assembled reads of ~ 1 in 333 bps AltHaplotype: 102 scaffolds (650 Kb, 50% with > 98% homology with main genome scaffolds) Repetitive scaffolds: 22 scaffolds (165 Kb) Excluded scaffolds: 39 scaffolds (28 Kb) Mitochondrion: 4 scaffolds (~79 Kb) 2420 out of 2494 (97.03%) JGI EST clusters mapped (90% ID 90% coverage) to the assembly J Schmutz et al.

JGI Melampsora portal will go public in November 2008 I Grigoriev & A Aerts

scaffold 212 scaffold 269 Althaplotypes 50% of Althaplotypes scaffolds present 98% homology with a larger scaffold in ‘main genome’ Althaplotype vs. main scaffolds (Blastn): Reconstruction of 10 scaffolds (75 kb)

Scaffold 5 Scaffold 6 Scaffold 212 Haplotypes/Althaplotypes Synteny WebACT: E Tisserand

29 largest Puccinia scaffolds vs 27 largest Melampsora scaffolds [Blastn] (~ 50 % of each genome)‏ ~ 15 syntenic blocks, inversions of 5 to 10 kb Synteny: Melampsora vs. Puccinia Supercontig 1 (Puccinia) vs Scaffold 10 (Melampsora)‏ WebACT: E Tisserand

tRNA tRNAScan-SE (default parameters, eukaryotic model) on all scaffolds. 253 tRNAs (24 in mitochondrion scaffolds), 194 contain an intron 49 out of 61 possible anti-codons tRNA are found as well as 2 tRNA for selenocysteine 10 tRNA pseudogenes, No tRNA suppressor # of tRNA detected in other basidiomycetes: L. bicolor: 279 C. neoformans: 141 P. chrysosporium: 200

Ribosomal DNA Melampsora larici-populina 5.8S ITS sequence & Puccinia graminis 28S and 18S sequences from NCBI used as query against all M. larici-populina scaffolds (blastn) rDNA tandem unit: 10.3 Kb Complete rDNA sequence used against M. larici-populina scaffolds: 22 complete copies There are 11 extra rDNA 18S and 20 extra rDNA 28S sequences elsewhere on other scaffolds Althaplotype: 460 : 1 Repetitive: Scaffolds # repetitions Main:

snRNA cmsearch (INFERNAL): 6 covariance models with cutoff & window size (RFAM)‏ parameters

Mitochondrial DNA Four scaffolds: sc146: 47,057 bp sc218: 18,446 bp sc332: 8,679 bp sc545: 4,301 bp Total78,483 bp